Diagnosis of tuberculosis and other diseases using exhaled breath

ABSTRACT

Disclosed are methods and devices for analyzing exhaled breath aerosols and exhaled breath condensates using various diagnostic tools that enable rapid, low cost and autonomous point of care assays for several diseases including respiratory tract diseases. Disclosed are methods and devices for analyzing exhaled breath aerosols and exhaled breath condensates for tuberculosis diagnosis using mass spectrometry, including MALDI-MS. The disclosed systems and methods provide for a diagnostic test result in less than about 20 minutes and provides for autonomous operation with minimal human intervention.

RELATED APPLICATIONS

This application is related to and claims the benefit of U.S.Provisional Application 62/891,954, filed Aug. 26, 2019, and entitled“Diagnosis of Tuberculosis and Other Diseases Using Exhaled Breath,” andU.S. Provisional Application 63/069,120, filed Aug. 23, 2020, andentitled “Diagnosis of Tuberculosis and Other Diseases Using ExhaledBreath,” which are both hereby incorporated by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

None.

FIELD

This disclosure relates to methods and devices for analyzing exhaledbreath aerosols and exhaled breath condensates using various diagnostictools to enable rapid, low cost and autonomous point of care assays forseveral diseases including respiratory tract diseases. Moreparticularly, but not by way of limitation, the present disclosurerelates to methods and devices for analyzing exhaled breath aerosols andexhaled breath condensates for tuberculosis diagnosis using massspectromtery, including MALDI-MS.

BACKGROUND

Tuberculosis (TB) has surpassed HIV/AIDS as a global killer with morethan 4000 daily deaths. (Patterson, B., et al., 2018). The rate ofdecline in incidence remains inadequate at a reported 1.5% per annum andit is unlikely that treatment alone will significantly reduce the burdenof disease. In communities with highly prevalent HIV, Mycobacteriumtuberculosis (Mtb) genotyping studies have found that recenttransmission, rather than reactivation, accounts for the majority (54%)of incident TB cases. The physical process of TB transmission remainspoorly understood and the application of new technologies to elucidatekey events in infectious aerosol production, release, and inhalation,has been slow. Empirical studies to characterize airborne infectiousparticles have been sparse. Two major difficulties plaguinginvestigation are the purportedly low concentrations of naturallyproduced Mtb particles, and the complication of environmental andpatient derived bacterial and fungal contamination of airborne samples.There have nonetheless been a number of attempts at airborne detection.A 2004 proof of concept study and subsequent feasibility study in Ugandasampled cough-generated aerosols from pulmonary TB patients. Coughingdirectly into a sampling chamber equipped with two viable cascadeimpactors resulted in positive cultures from more than a quarter ofparticipants despite their having received 1-6 days of chemotherapy. Afollow-up work employing the same apparatus found that participants withhigher aerosol bacillary loads could be linked to greater householdtransmission rates and development of disease findings which suggestthat quantitative airborne sampling may serve as a clinically relevantmeasure of infectivity. Therefore, interruption of transmission wouldlikely have a rapid, measurable impact on TB incidence.

The best method to control transmission of tuberculosis is to promptlyidentify and treat active TB cases. (Wood, R. C., et al., 2015).Diagnosis of pulmonary TB is usually done by microbiological,microscopic, or molecular analysis of patient sputum. The “goldstandard” test for TB infection in most of the developing world is asmear culture based on a sputum sample. The sample is smeared onto aculture plate, a stain is added that is specific to Mtb, and the stainedcells are counted using a microscope. If the concentration of cells inthe smear is greater than a set threshold, then the sample is classifiedas positive. If the TB counts are below this threshold, it is classifiedas negative. Diagnosis may take several hours. The need for sputum as adiagnostic sample is a limiting factor due to the challenges ofcollecting it from patients and to its complex composition. Theviscosity of the material restricts test sensitivity, increasessample-to-sample heterogeneity, and increases costs and labor associatedwith testing. Moreover, sputum production (which requires coughing) isan occupational hazard for healthcare workers. Sputum has severaldrawbacks as a sample medium. First, only about 50% of patients canprovide a good sputum sample. For example, children under about age ofeight often are not able to produce a sample upon request, usuallybecause they have not developed an ability to “cough up” sputum fromdeep in their throat. The elderly and ill may not have the strength tocough up sputum. Others simply may not have sputum in their throat.Thus, a diagnostic method based on sputum analysis may not provide adiagnosis in as many as 50% of the patients who provide a sputum sample.Sputum is also not useful as a diagnostic sample if it is collected oneto two days after a person has been treated with antibiotics because thesample is no longer representative of the disease state deep in thelungs, and within several days after treatment begins, the number oflive Mtb in the sputum is significantly reduced. Urine and blood havebeen proposed as sample media for the diagnosis of TB infection. Bloodis highly invasive and entails the higher cost of handling blood samplesthat are often HIV positive since, in some parts of the world, many TBpatents also have HIV co-infections. Further, a patient with an activeTB infection may not have many TB cells circulating in their blood.Urine-based diagnostics have also been proposed, but these tests lookfor biomarkers of the disease other than living TB bacilli, and none notbeen validated for widespread clinical use.

A sample that is easier, safer, and more uniform to collect and handlewould simplify TB diagnosis. Exhaled breath contains aerosols (“EBA”)and vapors that can be collected noninvasively and analyzed forcharacteristics to elucidate physiologic and pathologic processes in thelung. (Hunt, 2002). To capture the breath for assay, exhaled air ispassed through a condensing apparatus to produce an accumulation offluid that is referred to as exhaled breath condensate (“EBC”). Althoughpredominantly derived from water vapor, EBC has dissolved within itsnonvolatile compounds, including cytokines, lipids, surfactant, ions,oxidation products, and adenosine, histamine, acetylcholine, andserotonin. In addition, EBC traps potentially volatile water-solublecompounds, including ammonia, hydrogen peroxide, and ethanol, and othervolatile organic compounds. EBC has readily measurable pH. EBC containsaerosolized airway lining fluid and volatile compounds that providenoninvasive indications of ongoing biochemical and inflammatoryactivities in the lung. Rapid increase in interest in EBC has resultedfrom the recognition that in lung disease, EBC has measurablecharacteristics that can be used to differentiate between infected andhealthy individuals. These assays have provided evidence of airway andlung redox deviation, acid-base status, and degree and type ofinflammation in acute and chronic asthma, chronic obstructive pulmonarydisease, adult respiratory distress syndrome, occupational diseases, andcystic fibrosis. Characterized by uncertain and variable degrees ofdilution, EBC may not provide precise assessment of individual soluteconcentrations within native airway lining fluid. However, it canprovide useful information when concentrations differ substantiallybetween health and disease or are based on ratios of solutes found inthe sample.

Patterson et al. (2018) used a custom-built respiratory aerosol samplingchamber (RASC), a novel apparatus designed to optimize patient-derivedexhaled breath aerosol sampling, and to isolate and accumulaterespirable aerosol from a single patient. Environmental sampling detectsthe Mtb present after a period of ageing in the chamber air. 35 newlydiagnosed, GeneXpert (Cepheid, Inc., Sunnyvale, Calif.) sputum-positive,TB patients were monitored during one-hour confinement in the RASCchamber which has a volume of about 1.4 m³. The GeneXpert genetic assayis based on polymerase chain reaction (PCR) and may be used to analyze asample for TB diagnosis and to indicate whether or not there are drugresistance genes in the TB sample. The GeneXpert PCR assay for TB canaccept a sputum sample and provide a positive or negative result inabout one hour. The chamber incorporated aerodynamic particle sizedetection, viable and non-viable sampling devices, real-time CO₂monitoring, and cough sound-recording. Microbiological culture anddroplet digital polymerase chain reaction (ddPCR) were used to detectMtb in each of the bio-aerosol collection devices. Mtb was detected in77% of aerosol samples and 42% of samples were positive by mycobacterialculture and 92% were positive by ddPCR. A correlation was found betweencough rate and culturable bioaerosol. Mtb was detected on all viablecascade impactor stages with a peak at aerosol sizes 2.0-3.5 μm. Thissuggests a median of 0.09 CFU/litre of exhaled air for the aerosolculture positives and an estimated median concentration of 4.5×10⁷CFU/ml of exhaled particulate bio-aerosol. Mtb was detected inbioaerosols exhaled by a majority of the untreated TB-patients using theRASC chamber. Molecular detection was found to be more sensitive thatMtb culture on solid media.

Mtb can be identified in EBA by culture, ddPCR, electron microscopy,immunoassay, and cell staining (e.g., oramine and dmnTre). Of these, PCRand immunoassays have the potential to be rapid and specific to thespecies level. PCR and other genomics-based techniques can be specificto the strain level. Mass spectrometry has also been shown to bespecific to the strain level for cultures obtained from bacterialinfections. For example, the Biotyper from Bruker Daltonics (Germany),has been shown to be able to identify up to 15,000 strains of bacteriathat cause infections in humans. These techniques have been shown to becapable of identifying TB infection from EBA. Immunoassays for Mtbdetection, such as the one based on lipoarabinomannan, are also wellknown.

In the case of TB, people infected with TB are often diagnosed throughpassive case finding when individuals present themselves to clinics.Active case finding (“ACF”) is generally considered to include othermethods of reaching people suspected of TB infection outside of theprimary health care system. According to WHO, ACF is “systematicidentification of people with suspected active TB, using tests,examinations, or other procedures that can applied rapidly.” The goal ofACF is to get those infected to treatment earlier, reducing the averageperiod of infection, and thereby reducing the spread of the disease. Inthe case of TB, by the time an individual goes to a clinic for help,that person may have transmitted the TB infection to between about 10other people and about 115 other people. ACF can help to reduce orprevent significant TB transmission. The diagnostic systems and methodssuch as sputum analysis and blood analysis are either not automated andautonomously operated, or not rapid. Many have expensive assays that areconsumed for each analysis, and thus, do not have general utility foractive case finding, particularly in developing and under-developedcountries. As previously described, EBA analysis appears to be acompelling diagnostic tool for TB detection that provides for rapidanalysis, portability, and low cost because the need for expensiveassays and consumables are eliminated. McDevitt et al. (2013) havereport EBA analytical devices and methods for influenza diagnosis. Animpactor is used to remove large particles (>4 μm) from exhaled breath,followed by a wetted-film collector for the smaller particles (<4 μm).The two size bins of collected particles were analyzed for influenzavirus using a genomics-based method, reverse transcriptase polymerasechain reaction (rt-PCR). PCR technology uses biomolecular probes,combined with other biomolecules including enzymes, to amplify aspecific sequence of DNA if that particular sequence is present in thesample. The targeted sequences are believed to be specific to thedisease being identified. McDevitt et al. showed that EBA samples can beused to diagnose influenza. The disclosed devices and methods haveseveral shortcomings from a practical standpoint. First, the breathaerosol sample is collected into discrete samples that are severalmilliliters in volume, and thus, considerable effort is needed toconcentrate the sample. Further, the diagnostic device is not coupled toor integrated with the sample collector and is not amenable for use asan ACF tool. The ability to automate the RNA assays to create anautonomous diagnostic tool for TB analysis is not clear. A method todetermine whether sufficient volume of cough or breath aerosol wasgenerated by a particular patient is not described. As a result, if asample is found to be negative for influenza it may be due to a falsenegative resulting from inadequate sample collection. It is well knownthat there are large variabilities among humans with respect to thevolume of aerosolized lung fluid produced during various breathingmaneuvers.

The GeneXpert Ultra is a state-of-the-art genomics-based point of carediagnostic device which uses PCR technology. It may be integrated withan EBA sample collection method to perform ACF of TB and otherrespiratory diseases, but the sample collection times would be too longto be practical. Patterson et al. have shown that between 20 and 200 TBbacilli are typically produced in EBA and can be collected over aone-hour sampling period. A minimum of one hour of sampling would berequired to use the GeneXpert Ultra as a diagnostic assay. The GeneXpertmay be integrated with a system that samples air to analyze air samplesfor airborne pathogens. The BDS system (Northup Grumman, Edgewood, Md.),is being used for screening US Postal Service mail for bacterial sporesthat cause anthrax as the mail passes through distribution centers. Itcombines a wetted-wall cyclone with a GeneXpert PCR system toautonomously sample air and report if pathogens are present. However,the GeneXpert Ultra assay has a relatively high cost per test and takesapproximately an hour to complete the assay and provide a result. Ingeneral, PCR-based diagnostics are unsuitable for TB screening for ACFapplications due to both the extended time needed for sampling andanalysis, and the relatively high cost per test.

The time associated with a diagnostic assay is a critical parameter fora fielded, or “point of care” test. ACF is an example of a fieldeddiagnostic assay because, by definition, ACF takes place outside thehealthcare system. In the U.S., a point-of-care test needs to provide ananswer in 20 minutes or less. If not, the test is considered to be tooslow and not acceptable for achieving short patient wait-times. In thedeveloping world, and especially in countries with a history of TBprevalence, the GeneXpert may be used to provide diagnosis in about onehour. As previously described, this assay is expensive to implement on a“cost per test” basis, and therefore it is not yet widely deployed.Because of high cost, it is not used to screen patients who appearhealthy (non-symptomatic) but might have TB infection, but rather, isused to confirm a diagnosis that is strongly suspected based on othertests or factors.

Fennelly et al. (2004) described TB analysis using cough aerosol and acollection chamber that contains two Anderson cascade impactors usingindividuals who were known to have active patients. Individuals wereasked to provide two discrete five-minute bursts of intense coughing.Culturing of impacted samples took 30-60 days, and therefore thisapproach is not amenable to automation. A challenging aspect of EBA as aclinical sample is the relatively small sample of volume of exhaledparticulates that can be collected from breath. Further, a significantfraction of the mass collected is water. The molecules that containdiagnostic information (“biomarkers”) are present in nanoliter orpicogram quantities. Subsequently, the aerosol collection method must beeffective in capturing a large fraction of the biomass in the exhaledbreath. Exhaled breath includes air that is exhaled from the lungsthrough any number of maneuvers, including tidal breathing, deepbreathing, coughing, and sneezing. Particular types of deep breathingmaneuvers such as forced vital capacity (FVC), may be used to measurethe maximum volume of lung capacity by breathing in as much as possible,and exhaling as far (or as deep) as possible to maximize the volume ofexhaled breath. Forced expiratory volume (FEV) measures how much air aperson can exhale during a forced breath. The amount of air exhaled maybe measured during the first (FEV1), second (FEV2), and/or third seconds(FEV3) of the forced breath. Forced vital capacity (FVC) is the totalamount of air exhaled during an FEV test. Forced expiratory volume andforced vital capacity are lung function tests that are measured duringspirometry. Forced expiratory volume is an important measurement of lungfunction.

Although research has shown that respiratory diseases can be detectedfrom breath aerosol and breath condensate, modern clinical tests forinfections or diseases such as tuberculosis, influenza, pneumoniacontinue to utilize sputum, blood, or nasal swabs. Exhaled breathanalytical tools have not been commercialized because methods anddevices to efficiently collect and concentrate the trace amounts ofanalyte present in exhaled breath are lacking. Furthermore, there is nostandard or methodology to assess how much exhaled breath is sufficientfor a particular diagnosis. The disclosed exemplary devices and methodsovercome these limitations by collecting exhaled breath aerosol andbreath condensate at high flow rate, high efficiency, and intorelatively concentrated samples. Further, size sorting of aerosol can beincorporated to increase the signal to noise ratio for specific analytesprior to collection of the analytes. The concentrated samples may thenbe analyzed by several methods, but preferably, using methods that aresensitive, rapid, and highly specific to the analytes of interest. Morepreferably, the analysis will be rapid, and near real-time. Massspectrometry, real-time PCR, and immunoassays have the highest potentialto be sensitive, specific and nearly real-time.

A need exists for sample collection methods that can be coupled withfast diagnostic tools such as mass spectrometry (“MS”) that is morerapid and reliable than sputum analysis and less invasive than bloodanalysis to provide a diagnostic assay that is fast, sensitive, specificand preferably, characterized by low cost per test. Such a system couldbe used for active case finding (ACF) of TB and other lung orrespiratory tract diseases. To be effective, a system for ACF must berapid and inexpensive on a “per diagnosis” basis. Low cost-per-test is arequirement for screening a large number of individuals to proactivelyprevent TB transmission to search for the few that are indeed infectedTB. Low cost devices and methods would also be required forpoint-of-care diagnosis of influenza and other pathogenic virusesbecause patients probably infected with a “common cold” may be infectedwith rhinovirus. In some cases, the respiratory infection will be drivenby a bacterial or fungal microbe and may be treatable with antibiotics.In other cases, the microbe may be resistant to antibiotics, and adiagnostic method that can identify microbial resistance to antibioticsis preferable. Rapid EBA methods for distinguishing between viral andbacterial infections in the respiratory tract are desired whileminimizing the occurrence of false negatives due to an insufficientsample volume. Mass spectrometry, genomics methods including PCR, andimmunoassays have the highest potential to be sensitive and specific.Mass spectrometry, and in particular, MALDI time-of-flight massspectrometry (MALDI-TOFMS), is a preferred diagnostic tool for analysisEBA and EBC samples because it has been demonstrated to be sensitive,specific and near real-time.

BRIEF DISCLOSURE

Disclosed herein are exemplary methods and devices for EBA and EBCanalysis to provide a reliable diagnostic result, including TBdiagnosis, in less than 30 minutes, and preferably in less than 20minutes to enable point of care health services and minimize diseasetransmission using Active Case Finding. The devices and methods are alsocharacterized by low-cost on a per patient basis and are autonomous.

Disclosed is an exemplary autonomous system for diagnosis of respiratorydiseases in an individual using exhaled breath comprising a samplecollection subsystem comprising a sample extraction component configuredto receive an individual's face for extracting breath aerosol (EBA)particles expelled from the individual during a predetermined number ofbreath maneuvers into a flow of air fed into the extraction componentand a sample capture component fluidly connected to the sampleextraction component by an interface tubing and configured to separateand collect the EBA particles from exhaled breath and air as a collectedsample, and a sample analysis subsystem fluidly connected to the samplecapture component, the sample analysis subsystem comprising a sampleprocessing component to spot a small amount of the collected sample on asample plate and concentrate the collected sample on the sample plateand a diagnostic device for analyzing the sample. The EBA particles maycomprise at least one of microbes, virus, metabolite biomarkers, lipidbiomarkers, and proteomic biomarkers characteristic of the respiratorydisease. The flow rate of air entering the sample capture component maybe between about 100 L/min and about 1000 L/min. The flow rate of airentering the sample capture component may be between about 50 L/min andabout 500 L/min. The volume of the collected sample may be between about100 microliter and about 1 ml. The sample capture component may furthercomprise an air pump, and an impactor wherein the air pump provides theflow of air to carry the exhaled breath from the extraction componentinto the impactor and wherein the impactor separates the EBA particlesfrom exhaled breath to produce the collected sample. The impactor maycomprise at least one of a cyclone, a wetted wall cyclone, one or morewetted film impactors, and an impinger. The system may further compriseat least one virtual impaction stage disposed upstream of the impactor.The sample extraction component may comprise at least one of a coneshaped device, a shroud, CPR rescue mask, a CPAP mask, a ventilatormask, and a medical universal mouthpiece. The sample collectionsubsystem may further comprise a containment booth for receiving theindividual and isolating the individual's exhaled breath from theambient air wherein the extraction component is fluidly connected to thecapture component through a wall of the containment booth. Thediagnostic device comprises may comprise at least one of PCR, rt-PCR,immuno-based assay, mass spectrometer (MS), MALDI-MS, ESI-MS, GC-MS,GC-IMS and MALDI-TOFMS. The system may further comprise one or morechilling devices configured to be in thermal communication with thewalls of at least one of the interface tubing and the sample capturecomponent to chill the sample capture component. The sample capturecomponent may be chilled to a temperature greater than about 0° C. andless than about 10° C. using the one or more chilling devices. Thesample capture component may be chilled to a temperature greater thanabout 0° C. and less than about 4° C. using the one or more chillingdevices. The one or more chilling devices may comprise a Peltierthermoelectric device. The system may further comprise one or moresensors configured be in fluid communication with the sample extractioncomponent wherein the output of the one or more sensors is used tocalculate the total cumulative volume of exhaled breath aerosolparticles entering the sample capture component. The one or more sensorsmay comprise at least one of a CO₂ sensor, an oxygen sensor, a humiditysensor, an optical particle size counter, an aerodynamic particle sizer,and a nephelometer. The number of exhaled breath maneuvers required fordiagnosing respiratory diseases using the diagnostic device may bedetermined using the total cumulative volume of exhaled breath aerosolparticles.

The exemplary system may further comprise a sterilization component todisinfect the sample collection subsystem. The sterilization componentmay comprise at least one of a nebulizer for spraying a disinfectant,one or more UV lights to produce UV radiation, a steam generator, anozone generator, a peroxide vapor generator, and a combination thereof.The disinfectant may comprise at least one of 60% ethanol in water, atleast 60% isopropanol in water, and a peroxide solution. The collectedsample may be transferred to the sample processing component using atleast one of a dispensing pump, gravity-induced flow, and a roboticsample transfer system. The dispensing pump may be a peristaltic pump.The interface tubing may be made of at least one of copper, andnickel-copper alloy 400. The sample capture component may be made of atleast one of copper, and nickel-copper Alloy 400. The diagnostic devicemay comprise a MALDI-TOFMS. The sample processing component may furthercomprise at least one of a fluid reservoir, and a fluid dispensing pumpto dispense about 1 microliter of fluid on the collected sample disposedon the sample substrate. The fluid may comprise at least one of asolvent, a MALDI matrix chemical, water, and an acid. The individual maybe at least one of a person infected with at least one of tuberculosisand a corona virus disease, and a person who is not infected. Further,the lipid biomarkers may comprise biomarkers characteristic of Mtb. Thesample capture element may further comprise a packed bed column disposedin fluid communication with the sample extraction component toselectively capture EBA particles. The packed bed column may comprisesolid particles comprising at least one of resins, cellulose, silica,agarose, and hydrated Fe₃O₄ nanoparticles. The packed bed column maycomprise resin beads having C18 functional groups on the surface.

Disclosed is an autonomous method for diagnosing of respiratory diseasesin an individual using exhaled breath, comprising extracting EBAparticles expelled from the individual during a predetermined number ofbreath maneuvers into a flow of air fed into a sample extractioncomponent configured to receive an individual's face, collecting the EBAparticles from exhaled breath and air as a collected sample using asample capture component fluidly connected to the sample extractioncomponent by an interface tubing, spotting a small amount of thecollected sample on a sample plate, processing the sample by treatingthe sample using at least one of a solvent, a MALDI matrix chemical,water, and an acid, and mixtures thereof; and, analyzing the sampleusing a diagnostic device. The EBA particles may comprise at least oneof microbes, virus, metabolite biomarkers, lipid biomarkers, andproteomic biomarkers characteristic of the respiratory disease. Theprocessing step may further comprise concentrating the sample by dryingthe sample using suitable drying means. The diagnostic device maycomprise MALDI-TOFMS. The collecting step using the sample capturecomponent may comprise flowing the output from the extracting step intoa packed bed column to selectively capture the EBA particles andextracting the EBA particles from the packed bed column using at leastone of about 12.5% acetic acid, about 70% isopropanol, about 5% TFA,about 5% formic acid, and about 10% HCl in a sample extraction system toproduce a collected sample. The exemplary method may further comprisethe step of digesting the collected sample to generate a peptide samplecharacteristic of the EBA particles. The packed bed column may comprisesolid particles comprising at least one of resins, cellulose, silica,agarose, and hydrated Fe₃O₄ nanoparticles. The packed bed column maycomprise resin beads having C18 functional groups on the surface.

Disclosed is an autonomous method for diagnosing of respiratory diseasesin an individual using exhaled breath comprising instructing theindividual to position a sample extraction component for extracting EBAparticles from exhaled breath, initiating a predetermined set ofbreathing maneuvers to expel EBA particles from exhaled breath into aflow of air fed into the sample extraction component, flowing the EBAparticles in air into a sample capture component while chilling thewalls of the sample capture component and an interface tubing thatfluidly connects the sample extraction component and the sample capturecomponent, producing a collected sample, processing the collected samplecomprising the steps of treating the sample using at least one of asolvent, a MALDI matrix chemical, water, and an acid, and mixturesthereof, and analyzing the plated sample using MALDI-TOFMS. The flowrate of air entering the sample capture component may be between about100 L/min and about 1000 L/min. The flow rate of air entering the samplecapture component may be between about 50 L/min and about 500 L/min. Thevolume of the collected sample may be between about 100 microliter andabout 1 ml. The predetermined set of breath maneuvers may comprise thefollowing steps, performing a deep exhale to clear the individual'slungs, pausing for up to 10 s; performing an FVC inhale, performing adeep exhale, and, repeating the above sequence for up to 10 times. Theexemplary method may further comprise at least one of the steps of tidalbreathing, coughing, normal FVC breaths, speaking, and sneezing. Therespiratory diseases may comprise at least one of tuberculosis,influenza, pneumonia, cancer, and a disease caused by a corona virus.The number of pre-determined breath maneuvers may be determined by oneor more sensors that indicate at least one of the volume of particlesexhaled and the volume of breath exhaled. The one or more sensors maycomprise at least one of a CO₂ sensor, an oxygen sensor, a humiditysensor, an optical particle size counter, an aerodynamic particle sizer,and a nephelometer.

Disclosed is an exemplary system for diagnosis of a respiratory diseasecaused by aerosolized virus and bacteria particles, the systemcomprising a sample capture component to collect EBA particles in apredetermined volume of air into a flow of air fed into the samplecapture component as a collected sample wherein the air flow is betweenabout 30 L/min and about 1000 L/min and a sample analysis subsystemfluidly connected to the sample capture component, the sample analysissubsystem comprising a sample processing component to spot a smallamount of the collected sample on a sample plate and treat the collectedsample on the sample plate, and a diagnostic device for analyzing thesample. The sample processing component may comprise fluidic componentsto treat the sample using at least one of a solvent, a MALDI matrixchemical, water, and an acid, and mixtures thereof. The system mayfurther comprise one or more sensors configured be in fluidcommunication with the sample extraction component wherein the output ofthe one or more sensors is used to calculate the total cumulative volumeof exhaled breath aerosol particles entering the sample capturecomponent. The one or more sensors may further comprise at least one ofa CO₂ sensor, an oxygen sensor, a humidity sensor, an optical particlesize counter, an aerodynamic particle sizer, and a nephelometer. Thepredetermined volume of air may be determined using the output of theone or more sensors.

Other features and advantages of the present disclosure will be setforth, in part, in the descriptions which follow and the accompanyingdrawings, wherein the preferred aspects of the present disclosure aredescribed and shown, and in part, will become apparent to those skilledin the art upon examination of the following detailed description takenin conjunction with the accompanying drawings or may be learned bypractice of the present disclosure. The advantages of the presentdisclosure may be realized and attained by means of theinstrumentalities and combinations particularly pointed out in theappendant claims.

DRAWINGS

The foregoing aspects and many of the attendant advantages of thisdisclosure will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1. Schematic diagram of an exemplary EBA based diagnosis system.

FIG. 2. Schematic diagram of an exemplary EBA sample collectionsubsystem.

FIG. 3. Perspective view of a containment booth that may be optionallyused in the EBA sample collection subsystem.

FIGS. 4A-B. Volatile organic compound in exhaled breath analysis—ionchromatograms showing differences in spectral signatures between TB(FIG. 4A) and non-TB patients (FIG. 4B)

FIG. 5 is a schematic diagram of an exemplary diagnostic method usingexhaled breath aerosol (EBA) and exhaled breath condensate (EBC)analysis.

FIGS. 6A-C. Particle size distribution variability in exhaled breathfrom three healthy individuals using the modified FVC breathingmaneuvers.

FIG. 7. Carbon dioxide measurements in exhaled breath during variousbreathing maneuvers.

FIG. 8. Volume of lung fluid collected from exhaled breath duringdifferent breathing maneuvers.

FIG. 9. Weighted principal component analysis (PCA) of MS signalsacquired from positive and negative ion modes of TB and non-TB samples.

All reference numerals, designators and callouts in the figures arehereby incorporated by this reference as if fully set forth herein. Thefailure to number an element in a figure is not intended to waive anyrights. Unnumbered references may also be identified by alpha charactersin the figures and appendices.

The following detailed description includes references to theaccompanying drawings, which form a part of the detailed description.The drawings show, by way of illustration, specific embodiments in whichthe disclosed systems and methods may be practiced. These embodiments,which are to be understood as “examples” or “options,” are described inenough detail to enable those skilled in the art to practice the presentinvention. The embodiments may be combined, other embodiments may beutilized, or structural or logical changes may be made, withoutdeparting from the scope of the invention. The following detaileddescription is, therefore, not to be taken in a limiting sense and thescope of the invention is defined by the appended claims and their legalequivalents.

In this disclosure, aerosol generally means a suspension of particlesdispersed in air or gas. “Autonomous” diagnostic systems and methodsmean generating a diagnostic test result “with no or minimalintervention by a medical professional.” The U.S. FDA classifies medicaldevices based on the risks associated with the device and by evaluatingthe amount of regulation that provides a reasonable assurance of thedevice's safety and effectiveness. Devices are classified into one ofthree regulatory classes: class I, class II, or class III. Class Iincludes devices with the lowest risk and Class III includes those withthe greatest risk. All classes of devices as subject to GeneralControls. General Controls are the baseline requirements of the Food,Drug and Cosmetic (FD&C) Act that apply to all medical devices. In vitrodiagnostic products are those reagents, instruments, and systemsintended for use in diagnosis of disease or other conditions, includinga determination of the state of health, in order to cure, mitigate,treat, or prevent disease or its sequelae. Such products are intendedfor use in the collection, preparation, and examination of specimenstaken from the human body. The exemplary devices disclosed herein canoperate and produce a high-confidence result autonomously, andconsequently, has the potential to be regulated as a Class I device. Insome regions of the world with high burdens of TB infection, access tomedically trained personnel is very limited. An autonomous diagnosticsystem is preferred to one that is not autonomous.

The terms “a” or “an” are used to include one or more than one, and theterm “or” is used to refer to a nonexclusive “or” unless otherwiseindicated. In addition, it is to be understood that the phraseology orterminology employed herein, and not otherwise defined, is for thepurpose of description only and not of limitation. Unless otherwisespecified in this disclosure, for construing the scope of the term“about,” the error bounds associated with the values (dimensions,operating conditions etc.) disclosed is ±10% of the values indicated inthis disclosure. The error bounds associated with the values disclosedas percentages is ±1% of the percentages indicated. The word“substantially” used before a specific word includes the meanings“considerable in extent to that which is specified,” and “largely butnot wholly that which is specified.”

DETAILED DISCLOSURE

Particular aspects of the invention are described below in considerabledetail for the purpose for illustrating the compositions, andprinciples, and operations of the disclosed methods and systems.However, various modifications may be made, and the scope of theinvention is not limited to the exemplary aspects described.

An exemplary diagnosis system 100 based on exhaled breath analysis(“EBA”) may comprise an EBA sample collection subsystem 101, and ananalysis subsystem system 102 (FIGS. 1-3). These two subsystems aredescribed in detail below.

EBA Sample Collection Subsystem 101

Subsystem 101 may comprise sample extraction component 104 which may bein the form of at least one of a shroud, and a loose-fitting cone shapeddevice. A tight-fitting suitable mask suitable for receiving anindividual's face or that may be removably attached using straps and thelike to the face/head of a patient/individual 105 may also be used, butis not preferred because it is difficult to ensure a good fit on allhumans, especially men with beards/facial hair. The individual may sitin an optional containment booth 106 to isolate the patient's EBA fromthe ambient air in the testing room or area. An exemplary containmentbooth 106 may comprise a modified pulmonary function test body box assold by Morgan Scientific Inc. (Haverhill, Mass.) by replacing theplethysmograph components with the extraction component 104 describedherein such that booth 106 is in fluid communication with extractioncomponent 104. Booth 106 may also be a modified version of theRespiratory Aerosol Sampling Chamber (RASC) chamber described by Wood etal. (2016) and may incorporate the features and capabilities describedtherein. The disclosure of Wood et al. (2016), a non-patent literaturecited in the Reference section, is incorporated by reference herein inits entirety. In the RASC chamber, a participant is seated and engagespassively in an exhaled air sampling protocol. Approximately an hour isspent in the chamber following the phases outlined in Wood et al.Briefly, the chamber is sealed, and an air purge phase is performedentraining ambient air through high-efficiency particulate arrestance(HEPA) filters for a period of 10 minutes. This was followed by aparticipant-driven contamination phase in which the chamber is isolatedfrom the external environment and the proportion of exhaled air isallowed to rise to a 10% threshold defined by a chamber CO₂concentration of 4,000 ppm above the ambient level (based on an assumedexhaled air CO₂ concentration of 40,000 ppm). Measured CO₂ may be usedto calculate exhaled air volume as described in Wood et al. If thetarget is not reached after 30 minutes have elapsed, the sampling phaseis started at a lower exhaled air proportion. After sampling, thechamber is again purged to remove residual Mtb from the air.Contamination of the sampling chamber was driven primarily by tidalbreathing in addition to spontaneous coughing or sneezing. Particles andorganisms derived from sources other than breath were minimized by theparticipant wearing a full-body DuPont Tyvek suit during sampling and aninitial purge phase to minimize ambient contamination. Component 104serves to extract the aerosol particles emitted though the mouth andnose of patient 105 into a stream of air that acts as a sheath fluid(normally air) supplied from air source 107, which assists intransporting the aerosol toward the exit of component 104, and intosample capture component 108 without depositing on the walls ofcomponent 104. Air source 107 may be an air pump or compressor. Examplesof 104 are the funnel-shaped cone used by Milton's group or the facemask used by Fennelly. The air sheath fluid may be added though thewalls or at the large rim of the component 104, or more generally intothe booth 106. The air flow fed to component 104 may be suitablyfiltered (e.g., using a HEPA filter) to remove all or nearly allparticulate matter, including, but not limited to dust and fomites, inambient air. (HEPA, which stands for High Efficiency Particulate Air),is a designation used to describe filters that are able to trap 99.97percent of particles that are 0.3 microns, and is used to remove all ornearly all particulate matter, including, but not limited to dust andsoot, in ambient air. Further, the sheath fluid flow may be humidifiedto enable EBA particle size growth, thereby enabling a large fraction ofthe particles in the breath to be captured downstream in the aerosolcapture device 108. Interface tubing 109 fluidly connects extractioncomponent 104 to sample capture component 108 and may be further chilledto enable the EBA particles to grow in size. Chilling may be providedusing a refrigeration system or more preferably using Peltierthermoelectric cooling devices 113 that are small, lightweight andconsume less power to operate. A Peltier cooling device, for example, assupplied by Marlow Industries (Dallas, Tex.), TE Technologies Inc.(Traverse City, Mich.), generally comprises an array of alternating n-and p- type semiconductors. The array is soldered between two ceramicplates, electrically in series and thermally in parallel. Bismuthtelluride, antimony telluride, and bismuth selenide are the preferredmaterials for Peltier effect devices because they provide the bestperformance from 180 K to 400 K and can be made both n-type and p-type.Peltier effect creates a temperature difference by transferring heatbetween two electrical junctions when a voltage is applied across joinedalternating n- and p- type semiconductors to create an electric current.Heat is removed at one junction and cooling occurs. Heat is deposited atthe other junction and is readily removed with a fan or blower.

Component 104 may be disposable to limit the risk of a patient becomingcontaminated or infected with a pathogen emitted by a previous patient.Alternatively, component 104 may be reusable, in which case it may besterilized using sterilization component 110 using at least one of adisinfecting spray rinse produced using a suitable nebulizer, UVradiation, peroxide solution or vapor treatment, steam sterilization, ora combination thereof. A nebulizer, such as a Collision-type nebulizer(supplied by CH Technologies), may be fluidly connected near the exhaustof the cone, that is, near the throat region 111. A rinse fluid isnebulized to disinfect the extraction component 104, tubing 109 andcapture component 108. The rinse fluid is selected to ensure that theEBA and condensed EBA (exhaled breath condensate) samples and thecomponents in sampling subsystem 101 remains in a generally sterilecondition. For example, if 70% ethanol in isopropyl alcohol is used,this disinfectant solution may be readily removed from the sample byevaporation and does not interfere with the analysis. Sterilizationcomponent 110 (e.g., nebulizer) may be activated briefly at the end of asample collection period to provide a final rinse of the inlet tubing109 and capture component 108. Nebulizer 110 may again be activated toclean the sample extraction component 108 prior to the reuse by the nextpatient. Waste fluid may be pumped using pump 116 to a waste receptableby switching the valve 117 to fluidly connect pump 116 to the wastereceptacle. Although the exemplary sample extraction component 104 andsample capture component 108 may be disinfected between patients,exemplary system 100 with MS diagnostic devices do not require 100%decontamination of the exemplary systems between different individualsbecause of the high sensitivity to bioaerosol fragments of interest evenin the presence of trace contaminants.

Although extraction component 104 and tubing 109 is shown to beconverging/diverging in diameter, the diameter of the tubing may be thesame as the diameter of throat 111 of component 104 or may be larger orsmaller than the diameter throat 111. Sensors 112 include, but are notlimited to, a CO₂ sensor and a particle sizer/counter, and may befluidly connected to component 104 near throat region 111. Sensors 112provide an indication of the volume of exhaled breath that has beensampled. Continuous CO₂ monitoring allows for a close approximation ofthe proportion of exhaled air volume for each participant in thecontainment booth 106 at any given time. For example, for a person withreduced lung capacity, and having relatively small forced vital capacity(FVC), for example, less than two liters, or a weak cough, for example,less than 1 liter of exhaled breath, the patient may automatically, andin real-time be instructed to provide more FVC breaths or coughs until asufficient volume of exhaled breath aerosol has been collected. Wurie(2016) describes bioaerosol production by patients with tuberculosisduring normal tidal breathing and implications for transmission risk.Optical particle counter technology was used to measure aerosol size andconcentration in exhaled air (range 0.3-20 μm in diameter) during 15tidal breaths across four groups over time: healthy/uninfected,healthy/Mtb-infected, patients with extra-thoracic TB and patients withintrathoracic TB. High-particle production was defined as any 1-5 μmsized bioaerosol count above the median count among all participants(median count=2 counts/L). Data from 188 participants were obtainedpretreatment (baseline). Bioaerosol production varied considerablybetween individuals. Multivariable analysis showed intrathoracic TB wasassociated with a 3½-fold increase in odds of high production of 1-5 mmbioaerosols compared with healthy/uninfected individuals. Wurie (2016),a non-patent literature cited in the Reference section is incorporatedby reference herein in its entirety.

EBA sample capture component 108 may be a wetted wall cyclone (as shownin FIG. 2), one or more impactors (for example, as is demonstrated byMilton), or an impinger, that use dry or nearly-dry collection methodsfollowing by wash resulting in a resuspension of the EBA particles fromthe collection surface of the cyclone. Exemplary capture component 108includes, but is not limited to, wetted-film impactors (McDevitt, 2013),Coriolis™ wetted-wall cyclone (Bertin, France), impingers such as theBioSampler (SKC, Inc, Eight Four, PA), impaction-based devices such asthe BioCapture (FLIR Systems, OR), and 300 L/min wetted wall cyclone(King, 2012), and the BioSpot Sampler (Aerosol Devices, Fort Collins,Colo.). McDevitt's wetted film impactors and the BioSpot Sampler use acombination of humidification and condensation to “grow” the size of theaerosol particles, thereby enhancing the collection efficiency ofsubmicron-sized particles in EBA. Each of the non-patent literature andcontents therein published by McDevitt and King as cited in theReference section is incorporated by reference herein in their entirety.EBA aerosol particles may be collected directly into a liquid or may becollected on a filter medium and extracted by backflushing water orsolvent through the filter, using a dissolvable filter material anddissolving the filter, or pulverizing the filter in a liquid, and thenanalyzing the resulting slurry. EBA aerosol particles may be collectedby impacting the particles onto a dry surface and then washing theparticles from the surface with a suitable fluid. Virtual impactors maybe used to concentrate aerosols of a certain “cut size,” and thoselarger than the cut size. Virtual impactors, such as those described inU.S. Pat. No. 6,062,392, can be combined with impactors and otherdevices to increase the inlet air flow rate of air containing theexhaled breath as shown in U.S. Pat. No. 6,267,016. Sample capturecomponent 108 may include condensation growth tubes to grow submicronparticles into micron sized particles. Biomarkers may include lipidsfrom Mtb cell walls, and these lipids may be used in disease diagnosisin addition to Mtb cells. Close to 100% of the exhaled sample iscollected. There is no need to dilute the sample 115 collected withsaline solution.

EBA aerosol particle capture component 108 may have a flow rate sheathfluid (air) of between about 100 L/min and about 1000 L/min. The airflow rate is preferably more than 200 L/min and is about 300 L/min.McDevitt used flow rates of 130 L/min which is not sufficient to captureEBA produced during coughing reliably. The high flow rates minimize lossof aerosol due to blow-back during cough maneuvers. Higher flow rateslead to more entrainment of the EBA particles. Preferably, the aerosolcapture component 108 collects the particles into a small volume ofcondensed sample 115, and therefore produces concentrated aerosolbiomass (for example, at least 1 nanoliter of peripheral lung fluid perml of collection fluid). Sample 115 may be transferred to the analysissubsystem 102 using a pump 116, which is preferably a peristaltic pump.Valve 116 may be used to either route the condensed sample 115 to theanalysis subsystem 102 or to a waste receptacle, for example, duringdecontamination of sample collection system 101. The volume of EBAsample fluid of less than about 1 ml is preferred and targeted. Theexemplary disclosed system may be capable of and producing between about100 microliter and about 200 microliter of fluid. Therefore, not all ofthe exemplary EBA sample capture components as identified herein arepreferred for use in the disclosed exemplary sample collection subsystem101 for an autonomous system. For example, the BioSampler and Coriolisaerosol sampler collect EBA aerosol particles into aqueous samples thatare greater than 10 ml in volume. This large volume results in a verydilute sample, and a particle concentration method is needed. Apreferable aerosol capture component 108 would have high inlet air flowrate to entrain a large fraction of the particles in exhaled breath,even during a cough a sneeze flow rate of exhaled breath is very unevenin time. Similarly, McDevitt's wetted film impactor uses an injection ofsteam upstream of the impactor which is then condensed to providessamples that are collected into 50 ml centrifuge tubes, and thenconcentrated using a centrifuge. As previously described, the exemplaryEBA sample collection subsystem will capture particles in a liquidvolume that is about 1 ml or less. Similar to chilling tubing 109 thatfluidly connects extraction component 104 to component 108 using Peltierdevices, component 108 is preferably chilled using one or more Peltiercooling devices 114 to enable the EBA particles in the exhaled breath togrow in size. articles formed deep in the lungs may be on the order of100 microns in diameter but can be grown to greater than 1 micron. Thechilling of component 108 and tubing 109 facilitates condensation ofvolatile compounds in the exhaled breath which are also collected in theliquid sample. The exemplary EBA capture component 108 thereforecollects both volatile and non-volatile biomass in exhaled breath. Whena cyclone is used as capture component 108, the cyclone and the cycloneinlet tubing are preferably made of copper, copper alloys such as thenickel-copper Alloy 400 or other alloys having high thermal conductivityand low cost. Further copper and copper alloys have inherentantimicrobial properties. Peltier cooling devices 113 and 114 arepreferred as chilling devices due to the ease and accuracy with whichthey can control the temperature of the cyclone inlet 109 and the bodyof cyclone 108. Entrainment air fed into sample capture component 108(for example, when 108 is a cyclone) may be supplied using pump 118 andfiltered using HEPA filter 119.

EBA Sample Analysis Subsystem 102

EBA liquid sample 115 comprising EBA aerosol particles is then routed tosample processing component 120 for analysis using at least one of adiagnostic device 121 for analyzing EBA particles, and device 122 foranalyzing volatile organics in exhaled breath. Sample processingcomponent 120 may comprise elements necessary to perform one or more ofthe following steps:

(a) Sample 115 may be placed in a cup or vial. For example, the Series110A Spot Sampler (Aerosol Devices) uses 32 well plates with circularwell shape (75 μL well volume) or teardrop well shape (120 μL wellvolume) which are heated to evaporate the excess fluid/liquid in thesample to concentrate the sample.

(b) Sample 115 may be placed in a cup and exposed to a source of vacuumto cause the fluid to evaporate to concentrate the sample.

(c) Sample 115 may be mixed with a high volatility solvent (for example,methanol, ethanol, and acetonitrile) to accelerate the evaporativeprocess.

(d) Sample 115 may be subjected to a bead-based extraction. Bead basedextraction may be used to extract biomarkers from a dilute solution. Forexample, a micron sized magnetic bead may be coated with a glycanmaterial that binds well with protein biomarkers such as EBA particles.The beads may be intimately mixed with the EBA sample by an oscillatingmagnetic field. After a period of mixing, the beads may be pulled to oneside with a constant magnetic field, and then released into a smallvolume of solvent to extract the EBA particles as a concentrated sample.

(e) Sample 115 may be subjected to a solvent extraction process wherebythe sample is intimately contacted with an immiscible fluid such thatthe biomarkers (EBA particles) are preferentially transferred to theimmiscible fluid. For example, a relatively large aqueous collectionfluid sample (>1 ml) may be contacted with a relatively smaller volumeof organic solvent (for example, hexane or chloroform), transferringlipids from EBA particles and cell fragments in the sample to theorganic phase.

Many diagnostic devices may be adapted for use in analysis subsystem102, that include, but are not limited to devices that performgenomics-based assays (such as PCR, rt-PCR and whole genome sequencing),biomarker recognition assays (such as ELISA), and spectral analysis sucha mass spectrometry (MS). Of these diagnostic devices, MS is preferableon account of its speed of analysis. The MS techniques that arepreferable for biomarker identification are electrospray ionization(ESI) and matrix assisted laser desorption ionization (MALDI) MS. ESImay be coupled to high resolution mass spectrometers such as theOribtrap (ThermoFisher) ESI-MS devices are typically very large andheavy, and require a high level of expertise to operate, and are notsuitable for autonomous operation or applications such as point of carediagnostics. In contrast, MALDI-MS devices may be compact, lightweight,consume less than 100 watts of power and provide sample analysis in lessthan 15 minutes. MALDI-MS is a preferred diagnostic device forpoint-of-care diagnostics suitable for ACF. Including time for samplepreparation, the analysis time using MS may be less than about 15minutes. The sample must be dry before it is inserted into the MALDIspectrometer, and large (>1 ml) samples cannot be dried quickly withoutanalyte loss or degradation. With a concentrated sample 115, sampleanalysis using a MALDI MS may be less than 5 minutes (including thesample preparation) because less time is needed to evaporate the waterfrom the sample.

In “matrix assisted laser desorption ionization” (MALDI), largemolecules may be analyzed intact using mass spectrometry. In thistechnique, the target particle (analyte) is coated by a matrix chemical,which preferentially absorbs light (often ultraviolet wavelengths) froma laser. In the absence of the matrix, the biological molecules woulddecompose by pyrolysis when exposed to a laser beam in a massspectrometer. The matrix chemical also transfers charge to the vaporizedmolecules, creating ions that are then accelerated down a flight tube bythe electric field. Microbiology and proteomics have become majorapplication areas for mass spectrometry; examples include theidentification of bacteria, discovering chemical structures, andderiving protein functions. MALDI-MS has also been used for lipidprofiling of algae. During MALDI-MS, a liquid, usually comprised of anacid, such as tri-fluoro-acetic acid (TFA), and a MALDI matrix chemicalsuch as alpha-Cyano-4-hydroxycinnamic acid, is dissolved in a solventand added to the sample. Solvents include acetonitrile, water, ethanol,and acetone. TFA is normally added to suppress the influence of saltimpurities on the mass spectrum of the sample. Water enables hydrophilicproteins to dissolve, and acetonitrile enables the hydrophobic proteinsto dissolve. The MALDI matrix solution is spotted on to the sample on aMALDI plate to yield a uniform homogenous layer of MALDI matrix materialon the sample. The solvents vaporize, leaving only the recrystallizedmatrix with the sample spread through the matrix crystals. The acidpartially degrades the cell membrane of the sample making the proteinsavailable for ionization and analysis in an MS. Other MALDI matrixmaterials include 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),α-cyano-4-hydroxycinnamic acid (α-cyano or α-matrix) and2,5-dihydroxybenzoic acid (DHB) as described in U.S. Pat. No. 8,409,870.

In exemplary system 100, the use of chillers to chill the capturecomponent 108 and inlet line 109 produces sample 115 which comprisescondensed volatile organic compounds in exhaled breath and cooled liquidsample comprising EBA. Therefore, sample 115 may be routed to diagnosticdevices 122 to analyze condensed volatile organics and to device 121 toanalyze non-volatile EBA particles. During sample processing in step 204(FIG. 5), the liquid sample 115 may be warmed using a heater, driving ofthe volatile compounds into a diagnostic device 122 such as GC-MS,GC-IMS, volatile ion chromatography, or any other type of analysismethod suitable for analyzing volatile organic compounds. FIG. 4 showsthat that ion chromatograms from exhaled breath may be used todifferentiate between healthy individuals and TB-infected patients.Hashoul (2019) discloses use of sensors for detecting pulmonary diseasesincluding TB from exhaled breath. Hashoul describes breath analysis of226 symptomatic high-risk patients using GC (gas chromatography)-MS,pointing out several biomarkers of active pulmonary TB. They suggestedbiomarkers in oxidative stress products, such as alkanes and alkanederivatives, and volatile metabolites of Mycobacterium tuberculosis,such as cyclohexane and benzene derivatives. Their resultsdifferentiated between positive and negative TB with 85% overallaccuracy, 84% sensitivity and 64.7% specificity, using C-statisticvalues. Metal oxide sensors used. Moreover, the sensors array had asensitivity of 93.5% and a specificity of 85.3% in discriminatinghealthy controls from patients with TB, and a sensitivity of 76.5% andspecificity of 87.2% in identifying patients with TB within the entiretest population. The use of gold nanoparticle (GNP) and QMB (quartzmicrobalance sensors) were also discussed. The non-patent literaturepublished by Hashoul cited in the Reference section is incorporatedherein in its entirety. If chilling is not employed in subsystem 101 asdescribed above, liquid sample 115 may not contain any condensedvolatile organics. As a result, diagnostic device may be limited toanalysis of EBA particles in the liquid using device 121.

FIG. 5 is a schematic diagram of an exemplary diagnostic method 200using an exemplary system 100 as previously disclosed herein. Exemplarymethod 200 may be used to perform autonomous point-of-care diagnosisbased on exhaled breath. In step 201, the individual 105 may directed tobe seated; the chair may optionally be located in containment booth 106.In step 202, extraction component 104 may be removably fitted to theindividual's head or a cone that is larger than the head is positionedto fit loosely around the individual's head. Sample capture component108 is activated which causes air to be drawn around the patient's headand into the sample capture device. When a cyclone is used as samplecapture component 108 air flow to component 108 and chilling of thecyclone body and inlet tubing 109 are initiated. Preferably filteredsheath air is supplied to component 104. Sheath air may be humidified,preferably to greater than 90% relative humidity. Individual 105 is theninstructed to perform one or more predetermined maneuvers 203 which mayinclude a pre-set number of repetitions. The maneuvers may includeperforming one or more FVC or modified FVC maneuvers for generating EBAsamples from the lower respiratory tract, producing one or more coughsamples for generating EBA predominately from the upper respiratorytract, and producing one or more sneeze samples that generates EBApredominately from the nasal passages/upper respiratory tract. Amodified FVC is an FVC preceded by a deep exhale followed by a 5 to 10second pause. This exhale and pause cause bronchiole closure, followedby its reopening during the FVC inhalation. The closing and reopening ofthe small lung passages including the alveoli is believed to result inincreased particle production. For TB diagnosis using exemplary system100, between about 3 to about 5 modified FCV repetitions may be needed.A maneuver may include a coughing, FVC breathing, modified FVC breathingand sneezing. For diagnosis of some other diseases all maneuvers may beneeded. Sneezing may be induced by injecting a small dose of pepper orother spices in aerosol form into the nasal passages. Preferably, atleast three modified FVC breaths for sampling the lower respiratorytract, and a series of coughs for sampling the upper respiratory tractmay be needed. The lower respiratory tract generally comprises thetrachea, the lungs, and all segments of the bronchial tree (includingthe alveoli), and the organs of the lower respiratory tract are locatedinside the chest cavity. The upper respiratory tract generally comprisesthe nose, the pharynx, and the larynx, and the organs of the upperrespiratory tract are located outside the chest cavity. In some case, atleast ten modified FVC breaths may be needed. A series of sneezes may beinduced to sample the nasal passages. A preferred modified FVC thatincreases biomass collection per breath is one where the patient firstexhales, then pauses for up to 10 seconds prior to a FVC inhale,followed by a complete exhale as disclosed by Bake et al. (2019), anon-patent literature cited in the Reference section, which isincorporated by reference herein in its entirety. A preferable breathmaneuver may comprise 10× cough/FVC/bronchial blast. Instead of a coneor shroud, sample extraction 104 component may include continuouspositive airway pressure (CPAP) masks (e.g., Dreamware and Amara maskssold by Philips Respironics) that are used for treating sleep apnea.CPAP works by blowing air into the throat via a mask, subtly increasingair pressure in the throat and preventing the airway from narrowing butis modified to collect exhaled breath from various breath maneuversusing a flow of air as the sheath fluid.

The patient is then instructed to leave the chair and to be seated inthe waiting area. The extraction component 104 may be disinfected in UVlight. In sample processing step 204, sample 115 is automaticallytransferred from collection subsystem 101 for sample processing inanalysis subsystem 102. The type of sample processing depends on thetype of diagnostic device. When the diagnostic device is MALDI-MS,sample processing may comprise the steps of plating the sample 115 on toa MALDI-MS sample disk using a peristaltic pump, heating the disk toconcentrate the sample, adding the MALDI matrix/acid/solvent (describedbelow) and drying the disk. The sample disk is then analyzed using aMALDI-MS in step 205. The spectrum obtained is compared to spectra fromsamples that were known positives to specific respiratory infections,and also to spectra of samples form patients know to be healthy, and adiagnosis of the patient is generated. The result may then becommunicated to a clinician or to the patient. Once the extractioncomponent 204 is attached to the patient, and sample extraction isinitiated, the exemplary method is autonomous (with the exception ofasking the patient to the leave the chair) after performing the requiredmaneuvers and generates a test result of the diagnosis.

Regarding FVC maneuvers in step 203, FIGS. 6A-C shows the normalvariability of breath aerosol from healthy individuals for 10repetitions of the FVC breath maneuver. Even for health individuals, thevariability in the amount and particle size distribution of exhaledbreath aerosol is very large. The data were captured using a LASEX II(PMS, City, Colo.). A similar variability was also notice during EBAcollection from cough maneuvers. Particle size distributions help todetermine the total exhaled particle mass by integrating the particlesize distribution over time. This aspect helps to determine if thesample collected is sufficient for analysis. FIG. 7 shows the carbondioxide measurements in exhaled breath during bronchiole film burst(BB), FCV and cough maneuvers. A strong correlation was observed byPatterson et al. between CO₂ production rate and aerosol particleproduction. Measured CO₂ may be also used to calculate exhaled airvolume as described in Wood et al.

FIG. 8 shows the volume (nanoliters) of exhaled breath fluid volumebased on particle size distribution and particle concentrations asmeasured by the LASEX instrument. If the preferred method is the methodthat provides the largest sample, the modified FVC maneuver is preferredover standard FVC. Cough provides a similar amount of EBA sample volume,predominantly from the upper respiratory tract. Both of these methodsproduce sufficient sample to detect TB infection in patients otherwiseknown to be infected, but who are not yet on treatment.

Regarding the use of MS for detecting biomarkers for TB infection inexhaled breath, positive and negative ion signals containing 1000s offeatures were obtained (or extracted) using a high resolution Orbitrapmass spectrometer (ThermoFisher Scientific) from samples collected fromTB-patients (n=20), and non-TB/control samples (n=13). Masses above a5:1 signal to noise ratio (SNR) were selected. Weighted principalcomponents analysis (PCA), an unsupervised dimensionality-reductionalgorithm, was used to reduce the large set of signals to twocomponents. PCA provided 2-D visualization, which was used to explorewhether extracted signals would reveal intrinsic differences between twoclasses of samples, TB and non-TB. PCA results (FIG. 9) revealed thatthe samples of each group were prone to cluster together, suggestingextracted signals collected from high-resolution mass spectrometry couldbe used to distinguish between the two classes of samples.

The exemplary systems and methods described herein are not necessarilylimited in their diagnostic capability to respiratory infections. Lungcancer, for example, may also release biomarkers into the peripherallung fluid, and these biomarkers would be readily detected by thesystems and methods disclosed. Furthermore, because blood comes intointimate contact with the alveolar lining in the lungs, biomarkers ofinfection and cancer in other parts of the body (beyond the lungs) maybe transferred across the alveolar lining and into the peripheral lungfluid, and thus, may be detected by the analysis of EBA. As a result,the scope of the invention is not limited to the detection and diagnosisof respiratory diseases.

An exemplary sample extraction component may comprise a packed bedcolumn to selectively capture the non-volatile organic componentsincluding EBA particles in exhaled breath. The non-volatile componentsin exhaled breath may comprise breath aerosol particles comprising atleast one of microbes, virus, metabolite biomarkers, lipid biomarkers,and proteomic biomarkers characteristic of the respiratory disease. Thepacked bed column may comprise solid particles comprising at least oneof resins, cellulose, silica, agarose, and hydrated Fe3O4 nanoparticles.The packed bed column may comprise resin beads having octadecyl acrylate(C18) functional groups on the surface. The resin beads may have anominal diameter of between about 12 μm and about 20 μm. The solidparticles may comprise functional groups immobilized on the surface ofthe particles wherein the functional groups comprise at least one of C18(octadecyl), octyl, ethyl, cyclohexyl, phenyl, cyanopropyl, aminopropyl,2,3-dihydroxypropoxypropyl, trimethyl-aminopropyl, carboxypropyl,benzenesulfonic acid, propylsulfonic acid, an ion exchange phase, apolymer phase, antibodies, glycans, lipids, DNA and RNA. The ionexchange phase may comprise at least one of diethylaminoethyl cellulose,QAE Sephadex, Q sepharose, and carboxymethyl cellulose. The polymerphase comprises at least one of polystyrene-co-1,4-divinylbenzene,methacrylates, polyvinyl alcohol, starch, and agarose. The antibodiesmay comprise at least one of anti-human albumin, anti-Influenza A virusNP and Anti-SARS-CoV-2 virus. The antibodies may be immobilized onprotein A/G agarose beads. The capture element may be cooled to atemperature at or below ambient temperature. Commonly owned U.S.Provisional Pat. Appl. No. 63/069,029 titled “DIAGNOSIS OF RESPIRATORYDISEASES USING ANALYSIS OF EXHALED BREATH AND AEROSOLS,” providesadditional details related to exemplary sample extraction componentscomprising a packed bed column and is incorporated by reference hereinin its entirety.

The exemplary systems and methods disclosed herein may comprise roboticsystems and components. For example, the systems and methods maycomprise a robotic sample transfer system to spot a collected sample ona sample plate and conduct further processing or sample treatment, andanalysis of the treated sample. Commonly owned U.S. Provisional Pat.Appl. No. 62/931,200 titled “SYSTEMS AND METHODS OF RAPID AND AUTONOMOUSDETECTION OF AEROSOL PARTICLES,” provides additional details related toexemplary robotic sample transfer systems is incorporated by referenceherein in its entirety.

A collected sample need not be spotted on a sample plate prior toprocessing and analysis. For example, EBA particles in a collectedliquid sample may be aerosolized using a nebulizer and coated“on-the-fly” using a MALDI matrix to form coated aerosol EBA particles.The coated particles may be analyzed using aerosol time of flight massspectrometry (ATOFMS). “On-the-fly” means that the particles comprisingthe aerosol are not collected onto a surface (for example, onto thesurface of a MALDI plate) or into a liquid as a step in the coatingprocess. Commonly owned U.S. patent application Ser. No. 15/755,063titled “COATING OF AEROSOL PARTICLES USING AN ACOUSTIC COATER,” providesadditional details and is incorporated by reference herein in itsentirety.

The Abstract is provided to comply with 37 C.F.R. § 1.72(b), to allowthe reader to determine quickly from a cursory inspection the nature andgist of the technical disclosure. It should not be used to interpret orlimit the scope or meaning of the claims.

Although the present disclosure has been described in connection withthe preferred form of practicing it, those of ordinary skill in the artwill understand that many modifications can be made thereto withoutdeparting from the spirit of the present disclosure. Accordingly, it isnot intended that the scope of the disclosure in any way be limited bythe above description.

It should also be understood that a variety of changes may be madewithout departing from the essence of the disclosure. Such changes arealso implicitly included in the description. They still fall within thescope of this disclosure. It should be understood that this disclosureis intended to yield a patent covering numerous aspects of thedisclosure both independently and as an overall system and in bothmethod and apparatus modes.

Further, each of the various elements of the disclosure and claims mayalso be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of animplementation of any apparatus implementation, a method or processimplementation, or even merely a variation of any element of these.

Particularly, it should be understood that the words for each elementmay be expressed by equivalent apparatus terms or method terms—even ifonly the function or result is the same. Such equivalent, broader, oreven more generic terms should be considered to be encompassed in thedescription of each element or action. Such terms can be substitutedwhere desired to make explicit the implicitly broad coverage to whichthis disclosure is entitled. It should be understood that all actionsmay be expressed as a means for taking that action or as an elementwhich causes that action. Similarly, each physical element disclosedshould be understood to encompass a disclosure of the action which thatphysical element facilitates.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood asincorporated for each term and all definitions, alternative terms, andsynonyms such as contained in at least one of a standard technicaldictionary recognized by artisans and the Random House Webster'sUnabridged Dictionary, latest edition are hereby incorporated byreference.

Further, the use of the transitional phrase “comprising” is used tomaintain the “open-end” claims herein, according to traditional claiminterpretation. Thus, unless the context requires otherwise, it shouldbe understood that variations such as “comprises” or “comprising,” areintended to imply the inclusion of a stated element or step or group ofelements or steps, but not the exclusion of any other element or step orgroup of elements or steps. Such terms should be interpreted in theirmost expansive forms so as to afford the applicant the broadest coveragelegally permissible.

REFERENCES

-   1. B. Bake, P. Larsson, G. Ljungkvist, E. Ljungstrom, and A-C Olin,    “Exhaled particles and small airways,” Respiratory Research (2019)    20:8.-   2. Fennelly K. P., Martyny J. W., Fulton K. E., Orme I. M., Cave D.    M., et al. (2004) Cough-generated aerosols of Mycobacterium    tuberculosis: a new method to study infectiousness. Am J Respir Crit    Care Med 169: 604-609.-   3. Dina Hashoul and Hossam Haick, “Sensors for detecting pulmonary    diseases from exhaled breath,” Eur. Respir. Rev. 2019; 28: 190011.-   4. Hunt, J., “Exhaled breath condensate: An evolving tool for    noninvasive evaluation of lung disease,” J. Allergy Clin. Immunol.    2002; 110:28-34.-   5. Maria D. King, Andrew R. McFarland, “Bioaerosol Sampling with a    Wetted Wall Cyclone: Cell Culturability and DNA Integrity of    Escherichia coli Bacteria,” Aerosol Sci. Technol., 46:82-93, 2012.-   6. James J. McDevitt, Petros Koutrakis, Stephen T. Ferguson, Jack M.    Wolfson, M. Patricia Fabian, Marco Martins, Jovan Pantelic, and    Donald K. Milton, “Development and Performance Evaluation of an    Exhaled-Breath Bioaerosol Collector for Influenza Virus,” Aerosol    Sci. Technol. 2013 Jan. 1; 47(4): 444-451.-   7. Benjamin Patterson, Carl Morrow, Vinayak Singh, Atica Moosa,    Melitta Gqada, Jeremy Woodward, Valerie Mizrahi, Wayne Bryden,    Charles Call, Shwetak Patel, Digby Warner, Robin Wood, “Detection of    Mycobacterium tuberculosis bacilli in bio-aerosols from untreated TB    patients,” Gates Open Research 2018, 1:11.-   8. Wood R., Morrow C., Barry C. E., III, Bryden W. A., Call C. J.,    Hickey A. J., et al.: Real-Time Investigation of Tuberculosis    Transmission: Developing the Respiratory Aerosol Sampling Chamber    (RASC). PLoS One. 2016; 11(1): e0146658.-   9. Rachel C. Wood, Angelique K. Luabeya, Kris M. Weigel, Alicia K.    Wilbur, Lisa Jones-Engel, Mark Hatherill, and Gerard A. Cangelosi,    “Detection of Mycobacterium tuberculosis DNA on the oral mucosa of    tuberculosis patients,” Sci. Rep. 5, 8668 (2015).-   10. Fatima B. Wurie, Stephen D. Lawn, Helen Booth, Pam Sonnenberg,    Andrew C. Hayward, “Bioaerosol production by patients with    tuberculosis during normal tidal breathing: implications for    transmission risk,” Thorax 2016; 71: 549-554.

1. (canceled)
 2. The system of claim 51 wherein the EBA particlescomprises at least one of microbes, virus, metabolite biomarkers, lipidbiomarkers, and proteomic biomarkers characteristic of the respiratorydisease.
 3. (canceled)
 4. (canceled)
 5. The system of claim 51 whereinthe volume of the collected sample is between about 100 microliter andabout 1 ml.
 6. The system of claim 51 wherein the sample capturecomponent further comprises an air pump, and an impactor wherein the airpump provides the flow of air to carry the exhaled breath from theextraction component into the impactor and wherein the impactorseparates the EBA particles from exhaled breath to produce the collectedsample.
 7. The system of claim 6 wherein the impactor comprises at leastone of a cyclone, a wetted wall cyclone, one or more wetted filmimpactors, and an impinger.
 8. The system of claim 6 further comprisingat least one virtual impaction stage disposed upstream of the impactor.9. (canceled)
 10. (canceled)
 11. The system of claim 51 wherein thediagnostic device comprises at least one of PCR, rt-PCR, immuno-basedassay, mass spectrometer (MS), MALDI-MS, ESI-MS, GC-MS, GC-IMS andMALDI-TOFMS.
 12. (canceled)
 13. The system of claim 51 wherein thesample capture component is chilled to a temperature greater than about0° C. and less than about 10° C. using the one or more chilling devices.14-18. (canceled)
 19. The system of claim 51 further comprising asterilization component to disinfect the sample collection subsystem.20. The system of claim 19 wherein the sterilization component comprisesat least one of a nebulizer for spraying a disinfectant, one or more UVlights to produce UV radiation, a steam generator, an ozone generator, aperoxide vapor generator, and a combination thereof
 21. The system ofclaim 20 wherein the disinfectant comprises at least one of 60% ethanolin water, at least 60% isopropanol in water, and a peroxide solution.22. The system of claim 51 wherein the collected sample is transferredto the sample processing component using at least one of a dispensingpump, gravity-induced flow, and a robotic sample transfer system. 23-26.(canceled)
 27. The system of claim 51 wherein the sample processingcomponent further comprises at least one of a fluid reservoir, and afluid dispensing pump to dispense about 1 microliter of fluid on thecollected sample disposed on the sample substrate.
 28. The system ofclaim 27 wherein the liquid comprises at least one of a solvent, a MALDImatrix chemical, water, and an acid. 29-33. (canceled)
 34. An autonomousmethod for diagnosing of respiratory diseases in an individual usingexhaled breath, the method comprising: extracting EBA particles expelledfrom the individual during a predetermined number of breath maneuversinto a flow of air fed into a sample extraction component configured toreceive an individual's face; collecting the EBA particles from exhaledbreath and air as a collected sample using a sample capture componentfluidly connected to the sample extraction component by an interfacetubing; spotting a small amount of the collected sample on a sampleplate; processing the sample by treating the sample using at least oneof a solvent, a MALDI matrix chemical, water, and an acid, and mixturesthereof; and, analyzing the sample using a diagnostic device.
 35. Themethod of claim 34 wherein the EBA particles comprises at least one ofmicrobes, virus, metabolite biomarkers, lipid biomarkers, and proteomicbiomarkers characteristic of the respiratory disease.
 36. The method ofclaim 34 wherein the processing step further comprises concentrating thesample by drying the sample using suitable drying means.
 37. The methodof claim 34 wherein the diagnostic device comprises MALDI-TOFMS. 38.(canceled)
 39. The method of claim 34 further comprising the step ofdigesting the collected sample to generate a peptide samplecharacteristic of the EBA particles. 40-42. (canceled)
 43. The method ofclaim 34 wherein the flow rate of air entering the sample capturecomponent is between about 100 L/min and about 1000 L/min. 44-45.(canceled)
 46. The method of claim 34 wherein the predetermined numberof breath maneuvers comprises the following steps: a. performing a deepexhale to clear the individual's lungs; b. pausing for up to 10 s; c.performing an FVC inhale; d. performing a deep exhale; and, e. repeatingthe above sequence for up to 10 times.
 47. The method of claim 46further comprising at least one of the steps of tidal breathing,coughing, normal FVC breaths, speaking, and sneezing.
 48. (canceled) 49.The method of claim 34 wherein the number of pre-determined breathmaneuvers is determined by one or more sensors that indicate at leastone of the volume of particles exhaled and the volume of breath exhaled.50. The method of claim 49 wherein the one or more sensors comprises atleast one of a CO₂ sensor, an oxygen sensor, a humidity sensor, anoptical particle size counter, an aerodynamic particle sizer, and anephelometer.
 51. A system for diagnosis of a respiratory disease usingexhaled breath, the system comprising: a sample capture componentconfigured to collect EBA particles in a predetermined volume of air fedinto the sample capture component as a collected sample wherein the airflow is between about 30 L/min and about 1000 L/min; and, a sampleanalysis subsystem fluidly connected to the sample capture component,the sample analysis subsystem comprising: a sample processing componentto spot a small amount of the collected sample on a sample plate andtreat the collected sample on the sample plate; and, a diagnostic devicefor analyzing the sample.
 52. The system of claim 51 wherein the sampleprocessing component comprises fluidic components to treat the sampleusing at least one of a solvent, a MALDI matrix chemical, water, and anacid, and mixtures thereof.
 53. The system of claim 51 furthercomprising one or more sensors configured be in fluid communication withthe sample extraction component wherein the output of the one or moresensors is used to calculate the total cumulative volume of exhaledbreath aerosol particles entering the sample capture component.
 54. Thesystem of claim 53 wherein the one or more sensors comprises at leastone of a CO₂ sensor, an oxygen sensor, a humidity sensor, an opticalparticle size counter, an aerodynamic particle sizer, and anephelometer.
 55. The system of claim 51 wherein the predeterminedvolume of air is determined using the output of the one or more sensors.